Production of xanthan gum by fermenting a feedstock containing a mixture of mannose and glucose

ABSTRACT

A process of producing xanthan gum using a Xanthomonas microorganism is provided. Increased yields of xanthan gum are obtained by using mannose as part of the carbohydrate component of the fermentation feedstock. Preferably, the carbohydrate component of the feedstock is a mixture of mannose and glucose. More preferable is a mixture comprising 5 to 60% by weight mannose, particularly 20 to 45% by weight mannose, the balance being glucose although minor amounts of other sugars, e.g. maltose, may also be present. The carbohydrate component of the feedstock may be in the form of a raffinate stream from a glucose epimerization.

The present invention relates to a feedstock for a fermentationparticularly to a feedstock comprising mannose and more particularly tothe use of such a feedstock in a fermentation process for the productionof xanthan gum.

In the past fifty years fermentation processes have become of increasingsignificance for the production of complex organic molecules not capableof synthesis on an industrial scale by other means. Although theclassical fermentation processes have been known and used for centuriesto produce simple products such as ethanol and acetic acid it is onlyrelatively recently that microbial fermentation processes have beendeveloped to produce more complex products for the food, chemical andpharmaceutical industries.

A typical fermentation process involves the cultivation of a specialistmicroorganism on a suitable feedstock whereby the organism increases innumber and at the same time or subsequently produces metabolites some orall of which are desired products of the process. An example is thegrowth of Xanthomonas campestris on a suitable medium so as to producethe desired metabolite xanthan gum. Xanthan gum is a complexpolysaccharide containing D-glucose, D-mannose and D-glucuronic acidwhich has excellent rheological properties for applications in the food,pharmaceutical and chemical industries.

More recently, the range of microbial fermentation processes has beenextended by use of genetically engineered microorganisms and,additionally, fermentation processes have been devised in which theenzymes responsible for the fermentation are provided by a culture ofanimal cells, for example in the production of glycoproteins such as thehuman antihaemophilic Factor VIII.

The feedstock for a microbial or cell culture fermentation processgenerally comprises a carbohydrate, a source of nitrogen and variousminerals. The carbohydrate may be a complex substance such as starch ora maltodextrin which a microorganism for example can break down tosimple sugars before utilizing such sugars in its metabolic processes.More frequently however the fermentation feedstock is made up directlyfrom a simple sugar such as maltose or, more usually, glucose. It is forexample conventional practice to use glucose as the carbohydratecomponent in the Xanthomonas campestris fermentation referred to above.We have now found that in certain fermentation processes in which, as inxanthan gum, the desired product comprises mannose units, increasedyields of the desired metabolite may be obtained by using mannose aspart of the carbohydrate component of the fermentation feedstock.Mannose is an epimer of glucose but is less readily available and hencemore expensive than glucose. Despite the increased cost of mannosehowever we have found that it may be economic to replace part of theglucose conventionally used in a fermentation particularly if thereplacement is a less expensive mixture of mannose and glucose ashereinafter described because the increased yield of the, usually, highvalue product more than compensates for any increased cost of thefeedstock. All references to mannose in this specification are toD-mannose.

Accordingly, the invention comprises a fermentation process for theproduction of a product comprising mannose units in which a simple sugaris a component of the fermentation feedstock and which is characterizedin that part of the sugar is mannose.

Preferably the sugar component of the fermentation feedstock is amixture of mannose and glucose, more preferably a mixture comprising 5to 60% by weight mannose, particularly 20 to 45% by weight mannose, thebalance being mainly glucose although minor amounts of other sugars eg.maltose may also be present. Mannose may be produced by theepimerization of glucose in the presence of a catalyst eg. a molybdenumcompound. The product of this epimerization reaction is a mixture ofmannose and glucose, typically containing about 30% by weight mannoseand it is this mixture in particular which may be used as such as afermentation feedstock according to the present invention. It is alsopossible to use a glucose syrup as the epimerization feedstock eg. amixture of 90% or more glucose with glucose oligomers such as maltose inwhich case the fermentation feedstock will also contain small amounts ofthe glucose oligomers. The epimerization product may also be treated, egchromatographically, in order to separate mannose and a co-productraffinate stream comprising glucose and mannose. Such a raffinate streammay comprise 5 to 15% by weight mannose and may also be used asfeedstock in a process according to the present invention.Alternatively, if a desired fermentation product is relatively rich inmannose units it is possible to add mannose to the epimerization productor to enrich the product chromatographically to produce a suitablefermentation feedstock. The use of mannose/glucose mixtures isparticularly useful for the production of a fermentation productcomprising both mannose and glucose units.

The fermentation feedstock according to the invention is especiallyuseful in the production of xanthan gum by a fermentation processparticularly when the organism used is Xanthomonas campestris. As isshown in the Examples which follow in this specification, replacement ofpart of the glucose in such a fermentation by an equivalent amount ofmannose can give an improvement in yield of xanthan gum of more than 30%by weight.

The fermentation process according to the invention may be carried outbatchwise, in which case all of the feedstock is added at the start ofthe fermentation, or semi-continuously, in which the feedstock is addedprogressively throughout the fermentation, the mannose and anothersimple sugar such as glucose being added either or separately or,preferably, together.

The invention will now be further described and illustrated by referenceto the following Examples.

EXAMPLES 1 to 4

In the following fermentations the organism used was Xanthomonascampestris NRRL 1459-14 (LMG 574) type t1.

A pre-culture medium was used to prepare the innoculum for thefermentation. This medium had the following composition:

(all percentages in these Examples being by weight)

    ______________________________________                                        Malt extract      0.3%                                                        Yeast extract     0.3%                                                        Bacto-peptone     0.5%                                                        (nitrogen source)                                                             Dipotassium hydrogen                                                                            0.5%                                                        phosphate                                                                     Magnesium sulphate                                                                              0.02 %                                                      ______________________________________                                    

The solution was adjusted to pH 7 by addition of sulphuric acid beforebeing sterilized by heating at 120° C. for 20 minutes

A 20% carbohydrate stock solution which was separately sterilized byheating at 120° C. for 20 minutes was added to the above solution as a2% solution of either:

Glucose or A mixture of 30% mannose and 70% glucose

100 mls of the pre-culture media thus prepared were inoculated with theXanthomonas campestris organism from a Revco tube and incubated at 28°C. on a rotary table for 2 days.

The fermentation medium used in the Examples comprised:

    ______________________________________                                        Malt extract      0.3%                                                        Yeast extract     0.3%                                                        Bacto-peptone     0.5%                                                        Potassium dihydrogen                                                                            0.5%                                                        phosphate                                                                     Magnesium sulphate                                                                              0.02%                                                       ______________________________________                                    

This solution was adjusted to pH 7 before sterilization.

The carbohydrate components of the fermentation medium were glucose or a30% mannose/70% glucose mixture. The glucose or glucose/mannose mixturewas dissolved in 100 mls water to give concentrations of 1%, 1.5%, 2%and 3% respectively.

The fermenter had a 2 liter capacity and contained 1.3 liter of thesterilized medium described above plus, 100 mls of the glucose ormannose/glucose solution plus 100 mls of the pre-culture innoculum. Thetemperature was maintained at 29° C., the rate of aeration was 1.5 vvm(3 liters per minute) and the agitation rate was 850 rpm.

Each fermentation was allowed to continue for 18 hours after which timethe amount of xanthan gum produced was determined and the amount ofresidual sugars measured. The determination of xanthan gum was carriedout by diluting the fermentation medium with three times its volume ofisopropanol and filtering off the precipitated xanthan gum. Afterwashing the precipitate with two volumes of isopropanol, filtering anddrying in an oven at 40° C. overnight the xanthan gum was weighed.

Residual sugars were determined by high pressure liquid chromatographyof the medium after precipitation of the xanthan gum and evaporation ofthe isopropanol.

The results of the fermentations were as follows:

    __________________________________________________________________________                                Xanthan                                                                Xanthan                                                                              yield on                                                                            Xanthan                                                   Sugar(s)                                                                             concentration                                                                        sugar(s)                                                                            yield on                                    Example                                                                            Sugar(s) concentration                                                                        g/liter                                                                              consumed                                                                            total sugar(s)                              __________________________________________________________________________    1(a) glucose    1%   10.4   104   104                                         1(b) mannose/glucose                                                                          1%   11.6   119   116                                         2(a) glucose  1.5%   11.7    87   78                                          2(b) mannose/glucose                                                                        1.5%   14.0   102   93                                          3(a) glucose  2.0%   13.6         68                                          3(b) mannose/glucose                                                                        2.0%   14.5    91   72                                          4(a) glucose  3.0%   10.4         35                                          4(b) mannose/glucose                                                                        3.0%   13.9    88   46                                          __________________________________________________________________________

As can be seen from the results increases in yield were obtained in allthe four comparative pairs of experiments for the mannose/glucosemixture compared with glucose alone, the highest percentage increasebeing 34% in Example 4.

EXAMPLES 5 and 6

As in Examples 1 to 4 the organism used was Xanthomonas campestris NRLL1459-14 (LMG 574) type t1.

A pre-culture medium was used to prepare the innoculum for thefermentation. This medium had the following composition (all percentagesin these Examples being by weight):

    ______________________________________                                        Malt extract      0.3%                                                        Yeast extract     0.3%                                                        Bacto-peptone     0.5%                                                        (nitrogen source)                                                             Dipotassium hydrogen                                                                            0.5%                                                        phosphate                                                                     Magnesium sulphate                                                                              0.02%                                                       ______________________________________                                    

The solution was adjusted to pH 7 by addition of sulphuric acid beforebeing sterilized by heating at 120° C. for 20 minutes.

A 20% carbohydrate stock solution which was separately sterilised byheating at 120° C. for 20 minutes was added to the above solution as a2% solution of glucose.

100 mls of the pre-culture media thus prepared were inoculated with theXanthomonas campestris organism from a Petri plate and incubated at 28°C. on a rotary table for 4 days. The fermentation medium used in theExamples comprised:

    ______________________________________                                        Ammonium nitrate         1.144  g/l                                           Potassium dihydrogenphosphate                                                                          2.866  g/l                                           Magnesium chloride       0.507  g/l                                           Sodium sulphate          0.089  g/l                                           Citric acid monohydrate  2.3    g/l                                           Boric acid               0.006  g/l                                           Ferric chloride          0.024  g/l                                           Calcium carbonate        0.020  g/l                                           Zinc chloride            0.030  g/l                                           Concentrated hydrochloric acid                                                                         0.13   ml                                            ______________________________________                                    

This solution was adjusted to pH 7 with 5N sodium hydroxide solutionbefore sterilisation.

The carbohydrate components of the fermentation medium were glucose or a30% mannose/70% glucose mixture. The glucose or glucose/mannose mixturewas dissolved in 100 mls water to give a concentration of 3%.

The fermenter had a 2 liter capacity and contained 1.3 liter ofsterilized medium described above plus 100 mls of the glucose ormannose/glucose solution plus 100 mls of the pre-culture innoculum. Thetemperature was maintained at 29 ° C., the rate of aeration was 1.5 vvm(3 liters per minute) and the agitation rate was 850 rpm. During thefermentations the pH was controlled at 7 by the addition of a 2N aqueoussolution of potassium hydroxide. After 6.6 days fermentation when allthe sugars had been consumed the xanthan gum produced, was determined bydiluting the fermentation medium with three times its volume ofisopropanol and filtering off the precipitated xanthan gum. Afterwashing the precipitate with two volumes of isopropanol, filtering anddrying in an oven at 40° C. ovenight the xanthan gum was weighed.

The results of the fermentation were as follows:

    __________________________________________________________________________                   Xanthan                                                                              Xanthan                                                                concentration                                                                        yield on                                                                             Yield Stress*                                    Example                                                                             Sugar(s) g/l    total sugar(s)                                                                       Pa                                               __________________________________________________________________________    5(a)  glucose  9.78   32     20.5                                             5(b)  glucose/mannose                                                                        10.97  36     21.5                                             6(a)  glucose  10.5   35     18.5                                             6(b)  glucose/mannose                                                                        14.4   48     17.5                                             __________________________________________________________________________     *Yield stress is measured at 20° C. by a Bohlin CS Rheometer using     a 3% aqueous xanthan solution containing 0.1% sodium chloride.           

We claim:
 1. In a process for the production of xanthan gum whichcomprises fermenting a feedstock in the presence of a Xanthomonasmicroorganism which is effective to ferment said feedstock and formxanthan gum, the improvement in which the fermentation feedstockcomprises a mixture of mannose and glucose, said mixture comprising 5 to60% by weight mannose, the balance being glucose.
 2. A process as setforth in claim 1 in which the feedstock contains a minor amount of atleast one other sugar.
 3. A process as set forth in claim 1 in which themixture of mannose and glucose is the product obtained by theepimerization of glucose.
 4. A process as set forth in claim 3 in whichthe feedstock is a raffinate stream from a glucose epimerization andcontains 5 to 15% mannose.
 5. A process as set forth in claim 1 in whichthe microorganism is Xanthomonas campestris.
 6. A process for producingxanthan gum which comprises the step of fermenting a feedstock in thepresence of a Xanthomonas microorganism which is effective to fermentsaid feedstock and form xanthan gum, wherein the fermentation feedstockcomprises a mixture of mannose and glucose, said mixture comprising 20to 45% by weight of mannose, the balance being glucose.